| Background:Difficulty of nerve regeneration is a worldwide problem after spinal cord injury, at present which is considered that the inhibitory factors in central nervous system microenvironment are the main reason. Nogo, MAG and OMgp are now considered a kind of myelin inhibiting factors after spinal cord injury, which could be combined with NgR to inhibit the regeneration of axons and induce growth cone degeneration, and thus inhibit the repair of spinal cord injury. Among them, the Nogo-A inhibitory effect is the most obvious, which has become a hot research at home and abroad in view of the Nogo-NgR pathway in the repair of spinal cord injury.Traditional Chinese medicine "Jisuikang" as our clinical experience has the effect of analgesic, anti-inflammatory and promoting neurotrophic factor expression, which based on the previous research. However, how to promote the repair of spinal cord injury in "Jisuikang" is not clear. Therefore, the arm of the study by spinal cord injury preparated model in rats is to observe the dynamic expression of spinal cord tissue after spinal cord injury in Nogo-NgR path, and to explore the "Jisuikang" whether it can improve the microenvironment of the central nervous system through the intervention of Nogo-NgR signaling pathway, and whether it can promote nerve regeneration after spinal cord injury.Objective:To observe the dynamic expression of Nogo and NgR after spinal cord injury in rats according to the modified Allen’s model of spinal cord injury by immunohistochemistry, Western Blotting and fluorescence quantitative PCR, and to explore the significance of Nogo-NgR signal pathway in nerve regeneration process function. To observe the traditional Chinese medicine "Jisuikang" effect on spinal cord tissue pathology the tissue structure, cell apoptosis, GAP-43 protein and Nogo-A, NgR after spinal cord injury in rats, and explore its mechanism of action of promoting nerve regeneration after spinal cord injury.Methods:1.108 SD rats are randomly assigned into normal group (group N), sham operated group (group A) and model group (group B), with 36 rats in each group. Group N received no treatment, group A bites T10 spinous process and lamina only, avoiding injury the spinal cord, group B is made according to the modified Allen’s method. The rats are killed wthin 24h,3d,7d and 14d respectively after intervention(9 rats from each group).The expression changes of Nogo-A, NgR protein are detected in spinal cord of rats by immunohistochemistry and Western Blotting, to express the change of fluorescence quantitative PCR detection of Nogo-A, NgR mRNA.2.180 SD rats are randomly assigned into sham operated group (group A), model group (group B), prednisone group (group C), high dose of Jisuikang group (group D), middle dose group (group E), low dose group (group F), with 30 rats in each group. B, C, D, E and F groups are operated according to the modified Allen’s model of spinal cord injury method, group C are intragastric administration of prednisone 0.06g/(kg.d) after anesthesia recovery, group D for medicine "Jisuikang" 50g/(kg.d), group E for medicine "Jisuikang" 25g/(kg.d), group F for medicine "Jisuikang" 12.5g/(kg.d), and the same dose of saline are given by gastric lavage in group A and group B. Expression changes of rats were observed at 3d,7d and 14d by HE staining, transmission electron microscopy, apoptosis, GAP-43 protein and Nogo-A, NgR protein and mRNA.Results:1. Dynamic expression and significance of Nogo-NgR signal pathway in the spinal cord injury in rats.Immunohistochemistry, Western Blotting and fluorescence quantitative PCR show that the model group has the low protein and mRNA expression of Nogo-A and NgR at 24h, dropping to the lowest at 3 days, then rapidly peaking at 7 days, and gradually declining at 14 days after SCI, but still higher than that in sham operated group.2. The effect of structure and morphology on "Jisuikang" after spinal cord injury in rats.HE staining showes that model group appears obviously damage in spinal cord tissue structure of rats at 3 days after SCI, such as neuron pyknosis and necrosis, and the spinal cord tissue structure further destroys at 7 days, and visible infiltration of microglia, then at 14 days the number of glial cells in spinal cord tissue is increased than that of on the7th day, and inflammatory cell infiltrates. The prednisone group and Jisuikang group of high, low dose at each time point are lower than model group slightly in the structure of spinal cord injury, especially prednisone group and middle dose group obviously.Transmission electron microscope showes that the model group appeared on neuron membrane rupture, mitochondrial swelling, chromatin and nuclear fragmentation and structure disorder myelin at 3 days after SCI, then the damage progresses at 7 days after SCI. The prednisone group and Jisuikang high, low group on structure of spinal cord injury at each time point are lower than model group slightly, especially prednisone group and middle dose group obviously.3. The influence of "Jisuikang" on apoptosis and growth associated protein after spinal cord injury in rats.Immunohistochemistry of apoptosis showes that the number of apoptotic cells in model group are the most that at each time point after SCI (P< 0.01). Apoptotic cells is the least in prednisone group at 3 days after SCI (P< 0.05), the prednisone group and high dose group are beter than the other groups at 7 days after SCI, the effect of prednisone group and middle dose group are better at 14 days after SCI.Immunohistochemistry of GAP-43 showes that each group at each time point GAP-43 protein are different degree increased, and reaches a maximum at 7 days (P< 0.05). The expression of prednisone group and middle dose group are significantly higher than that of GAP-43 protein in model group at 3 days,7 days and 14 days (P< 0.01). The GAP-43 protein expression of high and low dose of Jisuikang group are significantly higher than that of the model group at 7 and 14 days (P< 0.05).4. Mechanism study of "Jisuikang" of regulation on the Nogo-NgR signaling pathway.Immunohistochemistry, Western Blotting and fluorescence quantitative PCR show that Nogo-A, NgR protein and mRNA expressions of spinal cord injury groups are relatively low at 3 days after SCI in rats, and the expression significantly increase on the 7th day, decreasing to some extent on the 14th day. Compared with the model group, Nogo-A, NgR protein and mRNA expressions of prednisone group and middle dose group are statistically significant at 3,7 and 14 days after SCI (P<0.05).Conclusion:1. The expression of Nogo-A, NgR protein in early once decreased after SCI in rats, then rapidly peaking at 7 days, and maintains a high level expression, which may be one of the important reason of nerve regeneration difficulty in central nervous system after spinal cord injury.2. "Jisuikang" could reduce the apoptosis of SCI cells, maintain the stability of lamellar structure of myelin, upregulate the expression of GAP-43 protein, and inhibit the expression of Nogo and NgR protein in the area of injured spinal cord. Among them, middle dose group was better than that of low and high dose group, and the effect is similar to prednisone group, which may be the mechanism of "Jisuikang" to improve the microenvironment of axonal regeneration, block the myelin sheaths of nerve regeneration inhibitory factor effect and promote the repair of spinal cord injury. |
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